1. Field of the Invention
The present invention relates to test devices that utilize a biochemical interaction between antigens and antibodies or fragments thereof to determine the presence of a target analyte. The invention also relates to methods for determining the presence of a target analyte in a biological fluid. More particularly, the invention relates to the use and activation of photoproteins to emit light in a localized region of a diagnostic assay device.
2. Description of the Related Art
Rapid and accurate determination of the presence or concentration of target analytes in biological fluids has drawn increasing attention in recent years. Enzyme Linked Immunosorbent Assay (ELISA) techniques employ an enzyme label which is used as a detection marker. Differing ELISA formats have been developed, but all employ an enzyme label, a detection means for determining the presence of the label, and a specific immunological binding reaction of an antigen-antibody pair. Although these assays can be somewhat simpler for laboratory technicians relative to other techniques for detecting the presence of an analyte species, these assays can be lengthy and can involve several steps and reagents. Accordingly, single step assays are desirable.
The traditional wet chemical method for detection of biological substances such as hormones, metabolites, ethical drugs, drugs of abuse, antibodies, and antigens, have, in some instances, been replaced by dry test strips which are conformed to generate a signal upon application of a test fluid containing target analyte. As will be appreciated, dry test strips are simple and convenient. However, the usual readout from such strips is a line or spots of color and not a light reaction. This is because previous assay devices have not provided a separate dry phase zone which can activate light generation by conjugated ligand that has reacted with analyte.
One recently developed dry strip method, disclosed in U.S. Pat. No. 4,446,232 to Liotta, hereby incorporated by reference, provides a technique for obtaining a rapid and reliable indication of analyte presence. Although satisfactory, there is still a need for an improved technique, particularly one which provides superior sensitivity of detecting the presence or concentration of analyte. Moreover, there is a need for an improved technique for signaling or indicating the presence or concentration of analyte.
The binding of ligand pairs, such as an analyte in a test sample and antibodies in a diagnostic assay, can be measured by labeling one member of the ligand pair with a photoprotein which produces light as a signal for detection. A bioluminescent protein particularly useful in labeling reagents for diagnostic assays is aequorin, as described in U.S. Pat. No. 5,486,455 to Stultz, hereby incorporated by reference. In the past, aequorin has been used to label antibodies and was found useful in standard format immunoassays in which aequorin labeled molecules were detected in solution by a luminometer. Photoproteins must be activated to emit light. Aequorin is activated by calcium ions, typically in the form of a solution of calcium acetate. For conventional tube luminometer measurements, the calcium acetate solution is squirted into a liquid solution containing an aequorin conjugated reagent. The addition of calcium acetate must take place in the dark so that ambient light does not reach the photomultiplier tube. Upon addition of calcium, the photoprotein flashes and the light is recorded by the luminometer.
The application of aequorin photoproteins in the prior art for diagnostic assays has been in the form of a kit utilizing several liquid reagents. The prior assay kits require multiple incubation and washing steps, introduction of liquid calcium solution in the dark, and quantitation of the signal by a tube luminometer. In this prior form, a light emitting analyte detection system could not be applied to rapid disposable diagnostic devices. Diagnostic assays in the prior art employing the activation of photoproteins require complicated multi-reagent kits, time consuming multiple steps, and quantitation by high cost luminometers. Accordingly, there is a need for an easy to use, one step, economical dry and disposable diagnostic assay that emits light to indicate the presence of an analyte.